Screening and monitoring assays are essential for the diagnosis and management of cancer. Cancer screening and monitoring tests, such as blood tests collected in a medical environment, can be useful for large scale screening of clinical healthy (or “asymptomatic”) individuals, for diagnosis, for prediction tests or for disease monitoring in subjects. Blood based remote samples for such applications have the advantage that it is convenient for a subject to provide a sample and the risk of side effects is extremely low. Therefore compliance is improved in a test population.
Clinically, tumors are defined as an abnormal mass. Solid tumors may be malignant tumors, consisting of malignant cells and a tumor stroma, thus being cancer. Different types of solid tumors include sarcomas derived from mesenchymal cells and carcinomas, derived from epithelial cells. The most common types of solid tumors are breast cancer, lung cancer, colorectal cancer and prostate cancer. Other solid tumors are gastric cancer, pancreatic cancer, hepatocellula, head and neck cancer, renal cell cancer, cancers of the female reproductive tract like ovarian cancer or endometrial or cervical cancer and germ cell tumors but also skin cancer including basal cell carcinoma, squamous cell carcinoma and malignant melanoma.
In the case of colorectal cancer (CRC), as a representative of solid tumors, only about 30% of people at risk are screened, partly due to psychological barriers induced by uncomfortable and invasive screening methods, such as colonoscopy.
However, CRC including colon cancer (i.e. large bowel cancer) and rectal cancer is the cause of over 655,000 deaths worldwide per year and represents the third most common form of cancer and the second leading cause of cancer-related death in the Western world. Typically, the diagnosis of colon cancer is made by colonoscopy in combination with a tissue biopsy. In loco regional disease, resection of the tumor and adjacent lymph nodes represents a standard of care. In colon cancer, surgery is followed by adjuvant chemotherapy in stage III disease (positive lymph nodes). In locally advanced rectal cancer, cT2/3, cN+ neoadjuvant radiochemotherapy is administered prior surgery.
As it is the case for various solid tumor entities, CRC can take many years to develop and an early detection of CRC significantly improves the chances of cure. For example, there are several different tests available for screening of CRC including digital rectal examination, the fecal occult blood test and various methods of endoscopy. As indicated before, colonoscopy represents the typical method of diagnosis. Moreover, certain biomarkers have been described which should be useful in the context of CRC, e.g. carcinoembryonic antigen (CEA) in the blood that is mainly used during follow up after curative or palliative treatment of CRC. Moreover, stool testing for occult blood and DNA are employed as screening methods for CRC. However, most of these tests do not allow differentiation between different stages of cancer.
For monitoring CRC, in particular during therapy, CEA may be used to monitor and assess response to treatment in patients with metastatic diseases. CEA can also be used to monitor the recurrence in patients post-operatively.
As in most solid tumors, colon cancer staging aims to determine the extent and distribution of disease (i.e. locoregional infiltration of the bowel wall and involvement of locoregional lymph nodes or presence and localization of distant metastasis).
It is performed during the diagnostic work-up and is a prerequisite to determine the optimal treatment strategy. Furthermore, staging is an important determinant of prognosis. The systems for staging CRC describe the extent of local invasion, the degree of lymph node involvement and whether there are distant metastases. For CRC, the most common staging system is the TNM (for tumors/nodes/metastases) system of the American Joint Committee on Cancer. The TNM system assigns a number based on three categories where “T” denotes the degree of invasion of the intestinal wall, “N” the degree of lymphatic node involvement, and “M” the degree of metastases. The broader stage of a cancer is usually quoted as a number—I, II, III, IV—derived from the TNM value grouped by prognosis; a higher number indicates a more advanced cancer and likely a worse outcome. The staging system is provided in the table below.
AJCC stageTNM stageTNM stage criteria for colorectal cancerStage 0T is N0 M0T is: Tumor confined to mucosa; cancer-in-situStage IT1 N0 M0T1: Tumor invades submucosaStage IT2 N0 M0T2: Tumor invades muscularis propriaStage II-AT3 N0 M0T3: Tumor invades subserosa or beyond (without other organs involvedStage II-BT4 N0 M0T4: Tumor invades adjacent organs or perforates the visceral peritoneumStage III-AT1-2 N1 M0N1: Metastases to 1 to 3 regional lymph nodes. T1 or T2.Stage III-BT3-4 N1 M0N1: Metastases to 1 to 3 regional lymph nodes. T3 or T4.Stage III-Cany T, N2 M0N2: Metastases to 4 or more regional lymph nodes. Any T.Stage IVany T, any N, M1M1: Distant metastases present. Any T, any N.
According to the present invention, the term “advanced colorectal cancer” refers to stage IV of the above table following the TNM staging system.
The appropriate treatment of a particular cancer depends on its TNM stage. If CRC is detected at an early stage, it can be curable. However, if it is detected at later stages (when distant metastases are present) it is less likely to be curable. Surgery remains the primary treatment while chemotherapy and/or radiotherapy may be recommended depending on the individual patient's staging and other medical factors. Recently, the overall survival time of patients with CRC, i.e. stage IV, has increased due to the development of novel therapeutic strategies combining chemotherapy, e.g. 5-fluorouracil, irinotecan, oxaliplatin, with e.g. antibodies against epidermal growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF), i.e., antibodies representing and acting as inhibitors of angiogenesis. However, these novel approaches are associated with increased toxicity and cost. Therefore, it is crucial to define biomarkers that can help identifying those patients who benefit most from such targeted therapies.
In colon cancer, chemotherapy after surgery is usually only given if the cancer has spread to the lymph nodes, i.e. at stages III or if distant metastasis is present, stage IV. Recently, it has been described that CRC patients that have a mutation in the k-ras gene do not respond to certain therapies, e.g. those that inhibit the epidermal growth factor receptor, e.g. cetuximab and panitumumab. Moreover, the b-raf oncogene as well as the k-ras oncogene have been utilised to predict outcome of EGFR antibody treatment in patients with metastatic CRC.
The VEGF antibody bevacizumab targets tumor angiogenesis. Presently, the first line chemotherapy regimens involve the combination of intravenous 5-fluorouracil, leucovorin and oxaliplatin with bevacizumab or alternatively, 5-fluorouracil, leucovorin and irinotecan with bevacizumab. The oral fluoropyrimidine capecitabin has been demonstrated to be equally effective as 5-fluorouracil, leucovorin and, therefore, combination chemotherapy with capecitabin and oxaliplatin or irinotecan is also widely used as a chemotherapy backbone. It has been demonstrated that a combination of chemotherapy plus bevacizumab significantly improves progression-free survival compared with chemotherapy alone and has become standard of care in the first and second line treatment of CRC (e.g. Saltz, L. B. et. al., 2008, J. Clin. Oncol. 26:2013-9). Clinical data indicate that bevacizumab without chemotherapy is not successful in treating solid tumors except for renal cell carcinomas (i.e. in combination with interferon-alpha) and possibly hepatocellular carcinoma.
However, in a recent phase III clinical trial response rates were comparable in both treatment arms (38% with and without bevacizumab, Saltz et. al. supra). Clinically, the identification of patients who are likely to respond to bevacizumab-containing treatment could be valuable particularly in patients who may undergo secondary resection of liver metastasis. While k-ras predicts the response for EGFR antibody treatment, equivalent biomarkers for bevacizumab in CRC patients are currently lacking.
The therapeutic blockade of VEGF by bevacizumab in CRC patients induces complex changes in a tumor stromal compartment including the loss of chaotic microvessels, remodeling of the vascular wall and a reduction in the interstitial fluid pressure. Such stromal alterations are part of the vascular “normalisation” process induced by bevacizumab and contribute to more efficient delivery of chemotherapeutic agents.
Angiopoietin-2 (Ang-2) is an inhibitory ligand of the Tie-2 receptor. The functional blockade of Tie-2 receptor tyrosine kinase by Ang-2 results in “abnormal” vessels due to the disruption of vascular wall integrity. In contrast to Ang-2, angiopoietin-1 (Ang-1) represents an agonistic ligand of Tie-2. Angiopoietins are glycoproteins that are involved in vascular development and angiogenesis. At present, four angiopoietins are known, named angiopoietin-1 to angiopoietin-4. Ang-2 is released in response to stimuli, such as injury, hypoxia and bacterial infection, and primes the endothelial activation response and promotes vascular permeability. Elevated Ang-2 levels have been described in patients with severe compared to mild sepsis. Elevated serum concentration levels of Ang-2 have been reported for patients with cancers other than CRC such as non-small cell line cancer and melanoma, where high serum Ang-2 levels are supposed to correlate with disease stage and poor survival (Park J. H., et. al., 2007, Chest 132:200-6; Helfrich I., et. al., 2009, Clin. Cancer Res. 15:1384-92).
Ochiumi, T., et al, 2004, Int. J. Oncol., 24, 539-547, suggest that Ang-2 expression in collaboration with VEGF expression at the deepest invasive site may be correlated with invasive/malignant potential and prognosis of advanced CRC. Both VEGF and Ang-2 are speculated as being correlated with tumor potential and prognosis. Holasch, J., et al., 1999-284, 1994-1998 describe vessel cooption, regression, and growth in tumors mediated by Angiopoietin and VEGF. It is speculated that Ang-2 may prove to be useful in the imaging of very small tumors and metastases.
In WO2006/069073 A2 the use of angiopoietins in anti-tumor therapy is described. A method for decreasing tumor growth is described wherein the amount of pericytes within vessels of the tumor is increased leading to a more mature status of the vessels, hence, improving the effects of the following radiation therapy.
In case of CRC, survival is directly related to the tumor stage at the time of detection. Survival rates for early tumor stages are about 5-times higher than for late stage cancers. Today, the CEA level is used to evaluate the response to surgical and non-surgical types of therapy. The CEA level is thought to correlate with the bulk of the tumor tissue. However, there are colorectal cancers that do not secret CEA into the blood stream.
Accordingly, there is a need in the art for approaches that afford detection and allow determination of the treatment regimen of solid tumors, in particular, CRC, having the added benefit of being cost effective, rapid, and minimally invasive, preferably non-invasive. Approaches that determine the prognosis of cancer and clinical outcome as well as allowing monitoring of treatment response and detecting relapse of cancers are of additional value and can help identifying those patients who benefit most from specific types of therapy as well as identifying the best therapeutic regimen for the individual patient.
There is also a need for allowing follow-up or monitoring the progression of CRC as well as for stratification of a subject with CRC, e.g. according to the treatment response of said subject using biomarkers, preferably by a non-invasive and cost effective method or system allowing the same.
For example, the combination of chemotherapy with the VEGF antibody bevacizumab has become standard in the first line treatment of metastatic CRC, however biomarkers predicting which patients are likely to benefit from these therapies remain elusive. It has already been demonstrated that tissue and plasma levels of VEGF in particular are not predictive of outcome in CRC patients treated with chemotherapy and bevacizumab.
The present invention aims for providing a new biomarker particularly useful in the issues described above.